| 1. Pick Colonies from plates made 07-21-2008 Start cultures.
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| 2. Plasmid prep: Prep RFP, YFP, GFP, TetR promoter, Terminator. Follow QIA miniprep procedure --> 1hr long.
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| 3. Spec the prep products: Using spectrophotometry, the DNA concentration of the plasmid prep products were measured.
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| 4. Double digest: Follow Kat's DNA work procedure to perform Double digest on: LacI Promoter, p22 cII gene, RFP, YFP, BV (dual promoters, GFP:Term already d.dig.). Incubate digested products for 2 hours @37C. Heat inactivate digestion enzyme for 15 mins @ 65C water bath. Follow the table below for double digest guidelines:
| Parts | 10x Buffer | BSA | H20 | DNA | RE 1 | RE 2
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| R2 | 5.0uL | 0.5uL | 40.5uL | 2.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| Y+2 | 5.0uL | 0.5uL | 40.5uL | 2.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| Y+4 | 5.0uL | 0.5uL | 39.5uL | 3.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| R+4 | 5.0uL | 0.5uL | 9.5uL | 33.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| Lac Pro. | 5.0uL | 0.5uL | 41.5uL | 1.0uL | 1.0uL, EcoRI | 1.0uL, Spe1
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| p22 cII | 5.0uL | 0.5uL | 39.5uL | 3.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| BV | 5.0uL | 0.5uL | 9.5uL | 33.0uL | 1.0uL, EcoRI | 1.0uL, Pst1
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| NOTE: RE stands for restriction enzyme. Parts chosen that had good 'spec' results, meaning there is a high concentration of DNA. R2 = RFP, Y+2 = YFP with LVA tag, Y+4 = YFP with LVA tag, R+4 = RFP with LVA tag, Lac Pro. = LacI repressed promoter, p22 cII = gene for p22 cII, BV = base vector. DNA added into each one correlates with which part is being used, for example: R2 would have 2.0uL of RFP, and Y+2 would have 2.0uL of YFP with LVA tag, etc..
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| 5. Vector Dephosphorylation: Same dephos. procedure used on GFP:Terminator sample and BV sample. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath. Follow the table below for guidelines:
| Parts | Antarctic phosphotase (enzyme) | Antarctic phosphotase Buffer
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| GFP:Terminator | Dephos. enzyme, 1.0uL | 5.6uL
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| BV | Dephos. enzyme, 1.0uL | 5.6uL
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| NOTE: Dephosphorylating enzyme and buffer were added into BV tube and GFP:Terminator tube, so no DNA had to be measured since was being put into that particular DNA tube already.
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| 6. Ligation Reactions: Procedure performed in 20 minutes. Once all were ligated, were then incubated @ 16C for 1 hour. Heat inactivated enzyme @ 65C for 15 minutes. Ligated the following using L4 DNA Ligase:
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| a. BV + TetR:p22 promoter + RFP
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| b. BV + TetR:p22 promoter + YFP
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| c. LacI:LambdacI + GFP:Terminator
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| d. LacI Promoter + p22 cII + BV
| Parts | 10x Buffer | H20 | BaseVector | Insert 1 | Insert 2 | T4 DNA ligase
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| (a) Lac/LAMBDAcI:GFP:term | 3.0uL | 6.0uL | 0 | 15.0uL Lac/LAMBDAcI | 5.0uL GFP:term | 1.0uL
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| (b) Lac/LAMBDAcI:GFP:term | 3.0uL | 6.0uL | 0 | 15.0uL Lac/LAMBDAcI | 5.0uL GFP:term | 1.0uL
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| LacIpro:p22cII:BV | 3.0uL | 15.5uL | 2.5uL | 0.5uL LacIpro. | 7.5uL p22cII | 1.0uL
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| Tet/p22:R2:BV | 3.0uL | 8.5uL | 2.5uL | 7.5uL Tet/p22 | 7.5uL RFP(R2) | 1.0uL
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| Tet/p22:Y+2:BV | 3.0uL | 8.5uL | 2.5uL | 7.5uL Tet/p22 | 7.5uL YFPw/LVAtag(Y+2) | 1.0uL
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| Tet/p22:Y+4:BV | 3.0uL | 8.5uL | 2.5uL | 7.5uL Tet/p22 | 7.5uL YFPw/LVAtag(Y+4) | 1.0uL
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| Tet/p22:R+4:BV | 3.0uL | 8.5uL | 2.5uL | 7.5uL Tet/p22 | 7.5uL RFPw/LVAtag(R+4) | 1.0uL
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| 7. Transformation: Procedure performed in 30 minutes. Transform all ligation products. Incubate in 2mL LB cultures for 2 hours @37C with shaking @ 220rpm's.
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| 8. Plate transformations: Plate transformation cultures.
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| 9. Prepare sequencing reactions: PROBLEM WITH SEQUENCING RESULTS. The ccdb gene still exists, but didn't terminate the E. Coli. This means the ccdb gene is either mutated or doesn't work properly. Solution: Pick 2 new base vector (DB3.1 E.Coli with ccdb gene) colonies in hope that the 2 new colonies will not have the same mutated/malfunctioned ccdb gene, and starting 2 new 50.0mL cultures to incubate O/N, from these cultures we will maxi-prep thus purifying out/extracting the DNA from the E. Coli and thus using this extracted plasmid/DNA to then use as the backbone.
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